1. Field of the Invention
The present invention relates to surface modification of polysaccharide, and in particular to mechanically embedding protein into the surface of polysaccharide to enhance cell attachment thereto.
2. Description of the Related Art
Mass cell production is important in tissue engineering, protein drug production, and cell therapy. Conventional mass cell production technology includes static flat culturing and dynamic bioreactor culturing. Static flat culturing is appropriate for small-scale trials such as 106-108 cells cultured in laboratory or factory. To compensate for the limited surface of the culture plate, large-scale cultures require numerous culture plates, however, manual operations such as seeding cells, changing media, passaging or harvesting cells increases labor burden and the risk of contamination. It is, therefore, not economical for mass production of animal cells.
Bioreactors provide sufficient metabolic exchanges and are popular in mass production of animal cells. Adherent cells cultured in bioreactor can be two- or three-dimensional. The former provides solid micro-beads for cells to attach to beads' surface with a two-dimensional manner and also evenly distributed nutrients. There are some drawbacks in this culture system. For example, cells will be dedifferentiation, much more shear stress effect, and friction is produced by stirring in the bioreactor. The latter provides porous matrices for cells to attach to porous contracture with a three-dimensional manner and provides more space for cell growth and less shear stress, although harvesting cells can be impeded. The proliferated cells may aggregate, and they will impede harvesting cells by regular digesting enzyme. For harvesting enough cell number, the longer digestion time is required, however, it will injure cells and reduce the recovery rate of healthy cells.
Most of mammalian cells are adherent, the growth of which includes attachment, propagation, and extension. Appropriate adherent materials may shorten attachment time for the cells to advance to log phase, increasing production efficiency and improving cell quality. Mass production of mammalian cells requires optimal media, appropriate mass transfer, low shear stress, and suitable carriers. Among these, cell carriers present the most critical issue. For long term cultivation of adherent cells in a bioreactor, various insoluble and porous carriers have been developed. These carriers are not designed to be implanted into living subjects and can be artificial polymers such as polystyrene (PS), polyvinyl chloride (PVC), or polymethyl acrylate resin. For example, U.S. Pat. No. 5,254,471 discloses a carrier for cell cultures comprising polyester fibers. The carrier makes it possible for the cells to retain their differentiation and proliferation ability for a long time, however, cell recovery from the carrier can be a problem. Carriers for cell recovery include two strategies. The cells can be embedded into a hydrogel and cultivation is performed in a hollow fiber bioreactor or a co-axial bioreactor. While cells can be recovered easily, the growth thereof may be suppressed by the low mass transfer of the carrier, imbedding long term cultivation. Cell recovery can be achieved by enzyme digestion such as trypsin digestion, but with inherent low recovery rate with high cell mortality. It is, therefore, important to develop a carrier having both insoluble and soluble properties for tissue engineering.
Developed carriers are mainly applied as implants or sustained-release carriers, rarely for cell cultivation. For example, U.S. Pat. No. 6,790,455 discloses a biodegradable and/or bioabsorbable fibrous matrix formed by biodegradable and/or bioabsorbable PLA/PLG/HA for delivering viable cells to a mammal using the cell storage and delivery system. U.S. Pat. No. 6,171,610 discloses a permeable, biocompatible support structure of PEO/PPO/PMA/PVA with a hydrogel-cell composition including a hydrogel, such as alginate, and tissue precursor cells. U.S. Pat. No. 6,656,508 discloses a sustained-release gel bead composition comprising PEG-alginate. U.S. Pat. Nos. 6,596,296 and 6,858,222 disclose bioabsorbable PLGA fiber for drug delivery. U.S. Pat. No. 6,103,269 discloses a thermoreversible sol/gel for releasing active agents. U.S. Pat. No. 6,471,993 discloses biocompatible PLLA/PGA/PEG/PMMA matrix for cell cultivation. U.S. Pat. Nos. 6,054,142 and 6,231,879 disclose a biocompatible cell device having an internal foam scaffold to provide a growth surface for encapsulated cells which produces a biologically active molecule. Most of these disclosures use insoluble cell matrix or gel for tissue transplant or drug release with no consideration for cell recovery.
A newly introduced matrix material is alginates, a family of unbranched polysaccharides with properties that vary widely depending on composition. For example, U.S. Pat. No. 4,614,794 (1986) discloses a complex of alginate and collagen for the formation of wound dressings and surgical implants. In addition, U.S. Pat. Nos. 5,529,914, 5,801,033, and 6,911,227 (1996) disclose a method for the formation of biocompatible membranes around biological materials using photopolymerization of water soluble molecules such as alginate or collagen, with no usage of cell culture. U.S. Pat. No. 6,306,169 (2001) discloses a biomechanical implant comprising collagen and a hydrated alginate gel, however, this implant is not for cell culture. U.S. Pat. Nos. 6,334,968, 6,425,918, and 6,793,675 (2002) disclose a method of forming polysaccharide sponges for cell culture and transplantation. A polysaccharide solution containing alginate is subjected to gelation to form a polysaccharide gel, and the gel is lyophilized to obtain a polysaccharide sponge.
The disclosed carriers still experience poor cell growth and low cell recovery rate, such that a need remains to develop a carrier which provides appropriate growth conditions and high cell recovery rate.